INTRODUCTION:

Pomalidomide is a chemical compound, 4-amino-2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione, a new immune modulatory drug (MiD), designed to be more potent and less toxic than thalidomide and lenalidomide ^,It is used in relapsed or refractory myeloma. The labs of Dr. Rober D'Amato led to the first report that 3-amino-thalidomide directly inhibits tumor cells and the vascular clusters of myeloma cancer. The HPLC methodsand other analytical methods have been reported for the conversion of Pomalidomide into a phosphate-buffered saline with human plasma in the μg/ml range^1-6. The UPLC-MS certified methods^7-13are reported for usage of negative ionization mode to detect Pomalidomide from rat plasma 9.998 to 1009.650ng/ml. LC-MS methods^14-17 were also published in the Pharmacokinetic study of Pomalidomide.

Since there is insufficient documentation to confirm Pomalidomide^18,19 in human plasma, employing UPLC/MS, this study has performed experimental validation, according to FDA guidelines.This method of validation was economical and could be used in pharmacokinetics of Pomalidomide.

MATERIALS AND METHODS:

Utilities and software:

The UPLC (Binary Solvent Manager, Waters) with autosampler (Sample Manager, Waters) was integrated with the Triple Quad Tandem weight spectrometer. The column oven was Acquity Waters. The chromatography and its integration were made by Mass Lynx version 4.1 software.

Reagents and Chemicals:

Pomalidomide and Celecoxib (IS) were purchased from Clearsynth Labs Limited, Mumbai, India., Acetone, acetonitrile, ammonium formate, formic acid, dimethyl sulfoxide and ethyl acetate were purchased from Merck Specialties Pvt. Ltd, Mumbai, India. Water was used from the purification systems (Milli Q, MilliPore, USA) and was stored in the lab. Human plasma lots (K2EDTA as anticoagulant) were obtained from Micro Therapeutic Research Labs Pvt. Limited, Chennai and used during the validation and analysis of the study sample. Plasma storage was at 70 ± 5°C.

Standards and their working solutions, Evaluation of standard solutions:

Pomalidomide and Celecoxib stock standard (IS) stock solutions were prepared with acetone. Further diluted with 80% acetone. The standard deviation of the eight concentration levels was adjusted by spiking the drug-free plasma with a stock solution of Pomalidomide to provide concentrations of 1.99, 5.75, 9.43, 18.86, 37.72, 75.44, 150.87 and 199.83ng/ml.

QC Standards:

LQC standards, MQC standards and HQC standards were made by spiking drug-free plasma and Pomalidomide to give solutions having 5.81, 74.97 and 149.95ng/ml concentrations respectively. They were stored at -20°C until analysis time.

Chromatography Conditions:

Chromatographic separation achieved by using Hypersil GOLD (50 mm × 4.6mm, 5µm), column. Mobile phase consists of 5mM ammonium formate buffer (pH 2.5) and acetone (20: 80v/v). Volume of injection has been 10μl. The rate of flow was 0.50ml/min. The total analysis time for one injection was 3.0 min. Column oven temperature was 40°C and autosampler temperature was 10°C.

Conditions of Mass Spectrometry:

The water Xevo TQ-S tandem system three times the liquid spectrophotometer with liquid electrospray ionization provides a good ionization mode. LC eluent was separated and approximately 0.100ml/min given into electrospray ionisation using the Ion Spray to produce positive ions at the interface. Mass spectrometric parameters are set as shown in Table 1.

Method for preparation of sample:

In 250µl plasma, 50µl ISTD (250ng/ml) was added along with 250µl 5mMm ammonium formate buffer and vortexed. The drug extraction process was carried out with 2.5ml TBME, centrifuged at 3500rpm/min for 5 minutes in a cooling centrifuge at 2-8°C. The 2 ml supernatant was pipetted out and evaporated in 40°C 15 psi of nitrogen until completely dry in evaporator. Reconstitution of residue was carried out with 400µl mobile phase and particular samples were labelled and made ready for column injection.

Validation Parameters:

Specificity of the method:

Solution having 2.004ng/ml (spiked LLOQ) has been injected to the column under optimised conditions of chromatography to indicate the Pomalidomide separation from any impurities or plasma. Selection was analysed for all the nine lots (6 blank K₂EDTA human plasma, 1 haemolysed plasma, 1 sodium heparin plasma and one lipedemic plasma) using 3 LCMS/MS instruments. The method specification was tested for plasma interference.

Linearity of the drug:

The spiked different concentrations were plotted counter to peak area ratios of the drug versus internal standard and best fitting line was calculated. The wide range of measurements was found out by solutions ranging from 1.990 to 199.838ng/ml.

Recovery studies of the drug:

The mean recovery percentages were found out by calculating the response of plasma that has been extracted. QC samples at high, medium and low against un-extracted QCsamples at high, medium and low were also calculated.

Precision and accuracy:

Inter-day precision and accuracy was evaluated by several repetition of analysis of plasma K₂EDTA samples containing different concentrations of Pomalidomide at different times. One run had a measurement of calibration curve, 6 replicates referring to quantitation lower limit and also lower, middle and high quality QC’s. Intra-day precision and accuracy was performed by analyzing the concentration of Pomalidomide at lower, middle and high quality QC’s and also at LLOQ.

Matrix effect:

Blank samples of plasma procured from 6 distinct human K₂EDTA plasma sources were subjected to processing and spiking with aqueous low and high QC’s. They were analysed along with dilute standard at every level of concentration. Effect of matrix on the ion suppression and enhancement were assessed by calculating the mean and% CV of IS normalization.

Stability studies:

Stability of stock solution of Pomalidomide and Internal Standard (Short term):

Pomalidomide solutions were prepared with acetone (Stability Samples) and stored at room temperature for 19.33 hours. Celecoxib solutions were prepared using diluent (acetone: water: 80:20) and stored for 19.25 hours at room temperature (stability samples). Analyte responses were considered to study the percentage stability in the given time.

Freeze-thaw stability:

Samples were made with low and high quality controls, frozen to -70°C. Some aliquots for quality control samples had to undergo four freeze-thaw cycles (stability samples). Quality control samples were prepared freshly (Comparison samples) and were processed along with replicates (six) of stability samples and analysed by a single run.

Stability of stock solution of Pomalidomide and internal Standard (Long term):

Pomalidomide solutions were prepared with methanol (Stability Samples) and stored in the refrigerator at 2-8° C for about 12 days. Celecoxib solutions were prepared with methanol (Stability Samples) and stored in the refrigerator at 2-8°C for about 12 days. Both stability and comparison samples have been diluted in the same concentration of the analyte and then analysed in the same run.

RESULTS AND DISCUSSION:

The chromatography detected during validation was represented as chromatograms. The established method was validated and confirmed for precision, accuracy, specificity, linearity, ruggedness and stability according to USFDA guidelines. The validation results are as given below.

Specificity:

Nine distinct plasma lots were used. Eight samples of LLOQ (9.999ng/ml) and plasma blank have been prepared from corresponding lots of plasma and were analysed. The results are as shown in Table 2. All the plasma blanks were analysed. The response detected at Pomalidomide retention time was lower than 20% of the LLOQ response, and at IS retention time, response was lower than 5% of the mean IS response in the LLOQ. The standard plasma blank chromatogram and LLOQ chromatogram are as shown in figures 2 and 3.

Linearity:

Calibration curve (ratio of peak area Versus Concentration) was found to be linear in the range of 1.9900 to 199.8380ng/ml concentration with 8 point calibration by using linear regression used for the quantification as shown in Figure 1. Regression values (r²) have been consistently higher than 0.99 in the process of validation of the method.

Recovery:

% Mean recovery for Pomalidomide in LQC, MQC and HQC was 50.68%, 53.43% and 57.49% respectively. The results are as shown in Table 3.

Accuracy and precision (Intraday and inter-day):

Coefficients of variation for within-run were ranging between 4.61% and 8.19% of Pomalidomide. The within run nominal concentration percentages were ranging between 90.80% and 113.04% of Pomalidomide. Results are as shown in table 5. Coefficients of variation for between-run were ranging between 8.38% and 9.32% of Pomalidomide. The between-run nominal concentration percentages were ranging between 100.52% and 104.46% of Pomalidomide. Results are as shown in Table 4.

Matrix effect:

The matrix effect of the analyte in percentage was 1.81% and 9.96% of Pomalidomide with low and high quality control samples.

Stability studies:

Stability of stock solution of Pomalidomide and Internal Standard (Short term):

Pomalidomide stability at LQC was found to be 100.73 % and HQC was 99.86%. The stability percentage of Celecoxib at LQC was 101.64% and at HQC was 98.99%.

Freeze-thaw stability:

Pomalidomide was stable in human K₂EDTA plasma even after 4 freeze-thaw cycles of both extremes - 70ºC ±15ºC and -30ºC±10ºC. The mean of nominal percentage of samples studied for stability at both the temperatures were 93.01 and 100.43% and 92.45 and 107.49%, respectively indicating stability after 4 freeze-thaw cycles.

Stability of stock solution of Pomalidomide and internal standard (Long term):

Pomalidomide is found to be stable in the refrigerator (2-8°C) and the percentage stability of Pomalidomide at LQC and HQC levels was 99.13 and 99.76%, respectively, indicating a stable solution for at least 12 days. The internal standard was found to be stable in refrigerator conditions (2-8°C) and the percentage stability of the internal standard at LQC and HQC level was 96.95 and 98.69%, indicating the stability for 12 days.

Figure 1: Calibration curve of Pomalidomide

Figure2: Pomalidomide and Celecoxib blank

Figure3: LLOQQC chromatogram sample for Pomalidomide

Table 2: Specificity and Selectivity for Pomalidomide and Celecoxib

PlasmalotID	Specificity(Blank)		Selectivity (SpikedLLOQ)		%InterferenceinBlank		AreaRatio	S/N Ratio(≥5)
PlasmalotID	Analyte	ISpeak	Analyte	ISpeak	Analyte(<20%)	IS(<5%)	Analyte/IS	Analyte
16-0512-II	307	961	6738	51824	4.5562	1.8544	0.1300	386.122
16-0513-II	334	911	4860	50677	6.8724	1.7977	0.0959	272.232
16-0514-II	325	817	4206	42097	7.7271	1.9408	0.0999	177.110
16-0515-II	336	804	6508	39010	5.1629	2.0610	0.1668	558.622
16-0518-II	180	393	4299	36027	4.1870	1.0908	0.1193	427.840
16-0519-II	148	408	7338	42433	2.0169	0.9615	0.1729	327.371
16-0071-X(H)	83	411	6032	43164	1.3760	0.9522	0.1397	482.546
16-0408-XI(L)	146	401	5946	44344	2.4554	0.9043	0.1341	476.673
16-0358-XIII(Hep)	127	383	6122	40512	2.0745	0.9454	0.1511	1020.567
				Mean	4.04760	1.38978	0.13441
						SD	0.026830
						%CV	19.96

(H)-Haemolyzed Plasma Lot, (L)-Lipemic Plasma Lot, (Hep)–Sodium Heparin Plasma Lot

Table 3: Recovery of Pomalidomide

Quality Control Samples ID	Aqueous IS Area	Extracted IS Area
Mean	34815.44444	36798.27780

Table 4: Inter-Batch or Total Precision and Accuracy of Pomalidomide

QCID	LOQQC	LQC	INTQC	MQC	HQC
Actual Concentration(ng/mL)	2.0040	5.8100	18.7440	74.9760	149.9500
Mean	2.05358	5.84025	18.73890	74.62401	156.63865
SD	0.292514	0.489212	1.508965	6.873977	14.602573
%CV	14.24	8.38	8.05	9.21	9.32
%Nominal	102.47	100.52	99.97	99.53	104.46

Table 5: Intra Batch Precision and Accuracy of Pomalidomide

QCID	LOQQC	LQC	INQC	MQC	HQC
Actual Concentration (ng/mL)	2.0040	5.8100	18.7440	74.9760	149.9500
Mean	2.08452	5.96397	18.29248	72.40245	145.99832
SD	0.123977	0.168487	0.525753	1.872981	11.108913
%CV	5.95	2.83	2.87	2.59	7.61
%Nominal	104.02	102.65	97.59	96.57	97.36

CONCLUSION:

A simple, fast, precise and accurate validated method of ultra liquid chromatography-tandem mass spectrophotometric (UPLC- MS/MS) was developed for the quantification of Pomalidomide and internal standard Celecoxib in human plasma. Chromatographic fraction was done using Hypersil gold column (50 mmX4.65mm, 5µ) with use of 5mM ammonium formate buffer (pH - 2.5) and acetone in 20: 80v/v (mobile phase). The rate of flow was around 0.50ml/min. The drug has been extracted using 2.5ml of tertiary butyl methyl ether (TBME). The specificity of this method was also checked for the plasma interference. The curve of calibration (ratio of peak area against Concentration) was linear over the concentration range of 1.9900 to 199.8380 ng/ml along with eight point calibration which is used for quantification by using linear regression. The % mean recovery for Pomalidomide in LQC, MQC and HQC was 44.59%, 51.78% and 58.10% respectively. The within-run coefficients of variation were ranging between 4.61% and 8.19% for Pomalidomide. The between-run coefficients of variation were ranging between 8.38% and 9.32% for Pomalidomide. The percentage matrix effect of analyte was found to be 1.81% and 9.96% for Pomalidomide for low and high-quality control samples. The long term and short term stability of Pomalidomide sample solutions and internal standard solutions were assessed. This method developed was also validated for the determination of Pomalidomide from plasma for other parameters namely Limit of Detection, Concomittant drug recovery, dilution integrity, ruggedness, carry over effect, reinjection reproducibility also and all the results confirm to acceptance criteria. The method is simple, fast, specific, highly sensitive, more accurate and more precise. Hence, the method can be successfully applied to a human pharmacokinetic study also.

ACKNOWLEDGEMENT:

The authors are grateful to Shri Vishnu College of Pharmacy, Bhimavaram and AU college of Pharmaceutical Sciences, Visakhapatnam for providing all the research amenities and I also extend my gratitude to MTR Research Laboratories, Chennai for providing all the research facilities.

CONFLICT OF INTERESTS:

Declared none.

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Received on 27.10.2021 Modified on 18.05.2022

Research J. Pharm. and Tech 2023; 16(4):1806-1810.

DOI: 10.52711/0974-360X.2023.00296

Parameters	Value
Capillary (kV)	3.00
Desolvation Gas (L/Hr)	1000
Cone Gas Flow (L/Hr)	150
DesolvationTemperature (°C)	450

Molecule	Parent (m/Z)	Daughter (m/Z)	Dwell (sec)	Cone (Volts)	Collision energy (eV)
Pomalidomide	274.43	201.1500	0.2	30	23
Celecoxib	382.12	362.03	0.2	30	30